uHTS identification of small molecule inhibitors of the mitochondrial permeability transition pore via an absorbance assay
Given its multifactorial roles, regulation of cellular Ca2+ metabolism and bioenergetics functions as an integrated system. In terms of normal physiology, this integration is reflected in mitochondrion's high capacity to store Ca2+ which may protect cells like neurons against transient elevation in intracellular Ca2+ during periods of hyperactivity. Furthermore, mitochondrial release of Ca2+ can more ..
BioActive Compounds: 5034
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1 R03 MH096534-01
Assay Provider: Michael Forte, Ph.D., Oregon Health & Science University, Portland, OR
Given its multifactorial roles, regulation of cellular Ca2+ metabolism and bioenergetics functions as an integrated system. In terms of normal physiology, this integration is reflected in mitochondrion's high capacity to store Ca2+ which may protect cells like neurons against transient elevation in intracellular Ca2+ during periods of hyperactivity. Furthermore, mitochondrial release of Ca2+ can amplify and sustain signals arising from elevation of cytoplasmic Ca2+ in response to extracellular events. An additional consequence of mitochondrial Ca2+ accumulation is the stimulation of oxidative metabolism through the activation of matrix Ca2+-sensitive dehydrogenases. As a result, mitochondrial Ca2+ homeostasis is tightly regulated and is based in a series of specific uptake and release systems. Importantly, regulation of ion fluxes across mitochondrial membranes, specifically the inner mitochondrial membrane (IMM), is essential since energy is stored in the form of a proton electrochemical potential difference which is used to drive both ATP synthesis and mitochondrial Ca2+ uptake and release.
One mitochondrial Ca2+ efflux pathway is represented by the mitochondrial permeability transition pore (mtPTP) which, in vitro, results in an IMM permeability increase to solutes with molecular masses of about 1,500 Da or lower. A great deal of information is available about the functional properties of the mtPTP.
In intact cells under normal conditions, mtPTP opens only transiently (and reversibly). These transient states likely mediate the fast release of Ca2+ from mitochondria in response to normal physiological signals that raise cytosolic, and hence mitochondrial Ca2+ levels to those required for mtPTP activation (the "threshold"). However, under pathological conditions, persistent activation of the mtPTP has dramatic consequences on cellular and mitochondrial function. This mode of activation results in the collapse of the membrane potential across the IMM (required to drive mitochondrial accumulation of Ca2+ and the synthesis of ATP) and depletion of pyridine nucleotides and respiratory substrates, causing respiratory inhibition and cell death. Consequently, mtPTP has long been implicated as a target for mitochondrial dysfunction in vivo, particularly in the context of specific human pathological events.
The goal of this high-throughput assay is to identify compounds that inhibit mtPTP. This is accomplished via the measurement of the change in absorbance of freshly isolated mitochondria in assay buffer due to swelling which occurs via the uptake of Ca2+ in the presence of test compounds.
1. Compounds are pre-spotted into assay plates the morning of or the night before the assay. Via the LabCyte Echo, 16 nL of 5 mM compound is transferred to Greiner, 1536-well, clear assay plates (Greiner 782101) to achieve 10 uM in 8 uL assay final volume. To the control wells in Columns 1-4, 16 nL of DMSO is transferred.
2. Prepare positive and negative control solutions, the mitochondrial suspension and the calcium solution working stocks according to the recipes in the Reagent Section.
3. Upon determination of activity, freshly isolated mitochondria from mice are suspended in assay buffer (Solution 1) and 4 uL of this solution is added to all wells of the assay plate with a MultiDrop Combi. Final assay concentration of mitochondria will be about 0.1 mg/mL (Working Stock ~0.2 mg/mL).
4. Following the addition of the mitochondrial suspension, 4 uL of the positive control working stock containing 2.0 mM EGTA in assay buffer (Solution 2) is added to Columns 1-2. Final assay concentration = 1.0 mM EGTA.
5. Next, 4 uL of Calcium solution (Solution 3) is added to negative control and test compound wells, Columns 3-48. Final concentration of calcium will be 40-100 uM (80-200 uM in the working stock).
6. Assay plate is immediately spun at 1000 rpm for ~60 seconds.
7. Plate is kept at room temperature for 30 minutes and then read on the BMG Pherastar utilizing absorbance at 540 nm.
Assay Buffer: 250 mM sucrose, 10 mM MOPS-Tris, 0.01 mM EGTA-Tris, 1.0 mM phosphoric acid, pH 7.4
Solution 1: 200 ug/mL mintochondria in assay buffer
Solution 2: 2.0 mM EGTA in Solution 3
Solution 3: 80-200 uM CaCl2 depending on mitochondrial activity, 5.0 mM glutamate, 2.5 mM malate in assay buffer Note: Concentration of calcium in the assay is dependent upon the activity of the isolated mitochondria which is determined via a calcium titration just before each high-throughput screening batch. A calcium concentration is used that allows for the arrival at a 2:1 window at the 30 minute time period.
Compounds that demonstrated a corrected % activity >= 50% compared to the controls are defined as active in the assay.
The experimental values were normalized by the difference between values from neutral and stimulator control wells in each plate. Then normalized data was corrected to remove systematic plate patterns due to artifacts such as dispensing tip issues etc. Further information about data correction is available at http://www.genedata.com/products/screener.html.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented.
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
** Test Concentration.
Data Table (Concise)